Our NICHD collaborators continue to develop theoretical models of light propagation in tissue, which account for scattering and absorption of light, and predict the spatial profile of the surface intensity of re-emitted and transmitted light. The model allows for both re-emission of the introduced light and fluorescent light generated by an embedded chromophore, either inherent to or introduced in the tissue. Measurement of a series of surface intensity profiles allows reconstruction of the three-dimensional structure of the tissue using inverse analytical techniques. Prototype instrumentation has been developed to capture surface images for analysis. A laser scanning system introduces light into the tissue at a series of sites in the region of interest and the emitted light is optically filtered through a dichroic filter and imaged onto a cooled charge coupled detector. Measurements using this instrumentation of fluorescent markers embedded in a highly scattering turbid medium yielded excellent agreement between the reconstructed theoretical prediction and the experimental measurement of these known sites. Identification of deeper structures within the tissue is possible by extending the instrumentation to capture images in the near infra-red region of the spectrum and by the use of novel infrared compounds to act as probes localized at desired sites within the tissue. This year we designed and fabricated several prototype optical instrumentation systems to support this work and is currently being evaluated for studying changes in the structure of cervical tissue. LBPS continues to explore , in collaboration with NCI, NICHD and within NIBIB, the use of nanocrystals as angiographic contrast agents and as substitute "dyes" for laser induced fluorescence diagnostic measurements. Upconverting nanocrystals - UPNCs - are now available, via a formal material transfer agreement, with sizes down to 10nm with surfaces modified to present amine or carboxylate groups as attachment agents for biological applications. A particularly appealing aspect of UPNCs is their potential use as diagnostic markers for the separation sciences such as capillary electrophoresis. A major limitation to the lowest level of detectability of diagnostic markers is the inherent fluorescent background signal from autofluorescence and/or the substrate/envelope. UPNCs potentially can reduce the background signal significantly using for example excitation source in the near ir, for example 980nm, and an emission in the visible, for example 550nm. This signal of record is widely separated from both any competing autofluorescence and the excitation laser source wavelength. In addition to the advantage this presents to the optical filtering requirements, many detectors for visible emissions are insensitive in the near infra-red. As an initial strategy to demonstrate effectiveness, a comparison will be made for an immuno-capture separation between UPNC and fluorescent dye tagged analytes. To this end an optical system using infra-red transmitting microscope objectives focuses the 980nm light on fused silica capillary containing the UPNCs. Orthogonal collection of the UPNC emission is achieved by either a close proximity optical fiber or a high numerical aperture objective each coupled, through a 550nm transmitting filter and 980nm rejection optical filters, to a photomultiplier detector. The use of polarized light has been explored in conjunction with NICHD and NCI as a method to track changes in tissue structure. Polarized photography offers the potential of distinguishing hidden structures developed below the skin surface, such as fibrosis resulting from X-ray radiation. A variable-angle polarized illumination system applied laser light to the surface of the skin of an athymic mouse. The scattered light (captured at an angle to minimize directly reflected light) was analyzed using a polarization sensitive detector for changes in polarization resulting from the interaction of the light and the tissue. Clear differences were observed for the degree of polarization between the skin of normal athymic mice and athymic mice irradiated with X-rays. Equi-intensity profiles of linearly polarized 650nm probe light diffusely reflected from skin and tissue-like phantom controls were fitted to ellipses. The orientation of the semi-major axis has a tendency to be perpendicular to collagen fiber orientation close to the entry point of the probe beam, but at larger distances the eccentricity becomes parallel to the fibers. Further, Fourier transform filtering of the polarization degree pattern allows the determination of the orientation and characteristic size of hidden structures developed under the skins surface under conditions such as fibrosis resulting from x-radiation. Fourier transform analysis of the polarization degree pattern and measurement of the equi-intensity profiles of a pencil-like polarized beam, backscattered from the skin, may allow characterization of fibrotic diseases, and may offer a means for safer radiation treatment. A key feature is the integration of the polarized illumination light into the polarization sensitive visualization optical axis.